Themis is a newly discovered protein regulating thymocyte development. It is very important in positive selection, but its function and mode of action are unknown. It is likely to be of importance to medicine as a single nucleotide polymorphism in THEMIS is strongly associated with celiac disease. Themis appears to act by altering the sensitivity of the T cell receptor to ligands that would normally be at the threshold between positive and negative selection. Themis alters TCR endocytosis and recycling. The hypothesis that this is how it mediates the modulation of TCR sensitivity will be tested. Alterations in the threshold between positive and negative selection regulated by Themis will be identified using thymus organ culture. Changes in signaling pathways caused by the loss of Themis will analyzed, and the subcellular localization of the Ras-Map kinase cascades in the Themis+ and Themis-deficient thymocytes will be determined. Themisdeficient thymocytes show altered TCR endocytosis, recycling and degradation. The mechanism of these changes will be identified, in terms of endocytic pathways, ubiquitination, and interactions with ArfGAP proteins. The important phosphorylation sites in Themis will be identified, and interactions with particular binding partners including the kinases Itk and ataxia telangiectasia mutated (ATM) will be dissected.